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(1) Field of the Invention
The present invention relates to an artificial insemination method which comprises providing sperm in a particle wherein the particle provides for the timed release of the sperm. In particular, a method for artificial insemination wherein the sperm in the particle is naturally or artificially capacitated after the artificial insemination. The present invention further relates to a method for encapsulating the sperm in particles and to the particles comprising sperm. In particular, the present invention relates to methods and particles wherein the sperm is provided in a semen extender that provides an energy source which does not facilitate capacitation of the sperm while in the particle. In a preferred embodiment, the particle comprising the sperm is a capsule or a bead.
(2) Description of Related Art
There is increasing demand for genetic transfer and using stored semen for artificial insemination as a means for genetically selecting animals with desirable traits such as production efficiency and meat quality. This demand has prompted a search for methods of storing semen that abrogates the decrease in viability of semen which occurs under prolonged storage. For example, the fertilizing ability of boar semen decreases during storage. Fertility of boar semen stored in liquid at 64xc2x0 F. in presently available extenders decreases from day one and is substantially diminished after 5 to 7 days. Thus, the farrowing percentages of sows inseminated artificially with semen after 5 days in storage are about half of the farrowing percentages of sows inseminated on the day of semen collection. The limited shelf life of boar semen not only restricts the shipping of semen samples to distant farms but also demands frequent deliveries, thus, the cost of artificial insemination is increased.
Another inherent problem of artificial insemination is the lack of a reliable method for determining the time of ovulation. Heat (standing estrus) detection allows the stockman to estimate when ovulation may occur. Females may be sexually receptive for 1-3 days, depending upon their age and previous experience. The natural estrous cycle in swine varies from individual to individual and ranges from 17 to 25 days. In addition, some sows may ovulate 12 to 24 hours before standing estrus; others may not ovulate until after estrus is detected. This inconsistent timing of ovulation in relation to standing heat further adds to the difficulties in determining when a sow should be inseminated. It is currently recommended that females be inseminated two or three times to compensate for inaccurate heat detection and variations in the timing of ovulation in relation to heat detection.
Currently, the most common method for storing semen is cryopreservation. However, thawing semen that has been frozen generally results in sperm damage.
Furthermore, cryopreservation also causes premature capacitation, which has been proven in bovine spermatozoa, thus, decreasing fertilization ability of the sperm (Cormier et al. J. Androl. 18: 461-468 (1997)). Therefore, there have been attempts to find other means for storing semen or for more effectively cryoprotecting the sperm.
Encapsulation of cells involving the polymerization of polyelectrolytes by multivalent ions is one of the most common methods for immobilizing whole cells. The first example of immobilizing a whole cell by this method was reported by Hackel et al. (Eur. J. Appl. Microbiol. 1: 291-293 (1975)). Currently, whole cell immobilization often uses ionically cross-linked alginate because it is considered non-toxic, economical, and simple to perform. Furthermore, alginate capsules can be coated with polylysine to increase the mechanical stability of the capsules.
U.S. Pat. No. 5,744,337 to Price et al. teaches using alginate to produce microspheres of controllable size in a method that involves internally controlling gelation of alginate in an oil:water emulsion. The method has been used to produce microspheres containing whole cells. The method allows the production of uniform spherical capsules less than 1,000 microns in diameter.
U.S. Pat. No. 5,912,005 to Lanza et al. teaches a method for implanting cells in a host using cells encapsulated in a temperature independent gel. The method uses ionically cross-linked alginate as the encapsulating material; however, to avoid having to use immunosuppressive chemicals to prevent host immune responses to the implanted capsules, the capsules are not coated with polylysine.
U.S. Pat. No. 5,846,530 to Soon-Shiong et al. teaches a method for encapsulating cells for transplantation wherein to strengthen the capsule, the polymer is covalently cross-linked. The covalently cross-linked capsules remain intact in the host for a longer period of time than tonically cross-linked capsules. The patent teaches covalently cross-linking lipids, polysaccharides such as alginate, and polycations to form capsules containing cells.
U.S. Pat. No. 5,084,350 to Chang et al. teaches a method for encapsulating cells consisting of encapsulating the cells in an ionically cross-linked alginate capsule, suspending the capsules in a solution and forming droplets containing one or more capsules, gelling the droplets, forming membranes on the droplets consisting of tonically cross-linked polylysine, and then liquefying the internal alginate capsules. The end product is a cross-linked polylysine capsule containing cells. The method prevents production of capsules containing cells that are trapped within the membrane.
U.S. Pat. No. 5,451,411 to Gumbotz et al. teaches using alginate capsules containing selected polyanions such as polyacrylic acid to shield the therapeutic agent therein from interaction with the alginate. For use in the host, the capsules are treated prior to use with an acid to reduce the molecular weight of the alginate and to prevent its interaction with the therapeutic agent.
Alginate has been used to encapsulate sperm cells. For example, Esbenshade and Nebel (Theriogenology, 33: 499-508 (1990)) encapsulated boar semen in capsules consisting of alginate capsules coated with polylysine. However, they found that the encapsulation caused an accelerated decrease in motility of the boar sperm. There was a complete loss of motility by 16 hours after encapsulation. The fertilizibility of the sperm was not evaluated. In another example, Nebel et al. (J. Anim. Sci. 60: 1631-1639 (1985)) encapsulated bovine sperm in polylysine coated alginate capsules; however, the sperm showed diminished motility and complete loss of fertility after encapsulation. In an effort to reduce spermatozoal injury of encapsulated sperm, Nebel et al. (Reprod. Fertil. Dev. 5: 701-712 (1993)) encapsulated sperm in polymers consisting of polylysine, polyvinylamine or protamine sulfate. The encapsulated sperm was capable of fertilization, but the encapsulated sperm were at a disadvantage to non-encapsulated sperm when cows were inseminated at conventional times.
Another approach to encapsulating sperm was the use of a thermal gel, wherein the thermal gel was solid at room temperature and liquid at body temperature. U.S. Pat. No. RE34,326 to van Blerkom teaches a method for encapsulating sperm for artificial insemination in nontoxic polymers such as polyurethane-polyether polymers which are freely flowing at body temperature and a gel or solid at room temperature. Bovine sperm encapsulated in the polymer and stored for 14 days at 7xc2x0 C. retained about 48% of its motility. Fertility of the sperm was not determined.
Therefore, while the prior art has taught methods for encapsulating semen, the prior art has not provided a proven means for prolonging the shelf life of liquid semen or reducing the damage to frozen semen. Thus, there remains a need to prolong the shelf life of semen, particularly the shelf life of boar semen. There is also a need for a method to extend the fertile period of the sperm cells in the female reproductive tract. Prolonging the shelf life of the semen would decrease the cost of the semen and the costs for shipping, it would improve the breeding efficiency by increasing the number of females settled by artificial insemination, it would decrease the labor involved in artificial insemination by reducing the number of times artificial insemination needed to be performed to achieve maximum pregnancy, and it would improve overall artificial insemination management because the variation in the timing of ovulation becomes less critical for successful artificial insemination.
The present invention comprises a method for encapsulating sperm within a particle comprising the steps of: (a) providing a sperm sample comprising a semen extender which provides an energy source that does not facilitate capacitation of the sperm and a gel forming polymer; (b) forming the sperm sample into a droplet containing the sperm sample; and (c) forming the particle by inducing the gel forming polymer to gel. Preferably, the semen extender is substantially free of glucose and reactive oxygen species and provides an exogenous energy source which does not facilitate capacitation of the sperm in the absence of the reactive oxygen species or the glucose. Preferred exogenous energy sources include those selected from the group consisting of fructose, fructose-6-phosphate, pyruvate, lactate, and mixture thereof.
In a particular method of the invention, the particle is a capsule comprising the sperm sample encapsulated by a membrane comprising the gel forming polymer wherein the gel forming polymer, preferably an ionotropic polymer, is induced to gel by a polyvalent ion. The gel forming polymer can be selected from the group consisting of sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, gelatin, iragacanth gum, xanthan gum, and mixture thereof.
In another method of the invention, the particle is a bead comprising the sperm sample encapsulated in a thermal gel that is a solid at room temperature and a liquid at body temperature. Preferred thermal gels include those selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, and mixture thereof.
The present invention also provides several particular methods for encapsulating sperm within a capsule. The first method comprises the steps of (a) providing a sperm sample which is in a semen extender and a gelling inducer; (b) forming the sperm sample into a droplet containing the sperm sample, the droplet having an outer surface; (c) providing a gelling solution comprising an effective amount of a gel forming polymer which polymer gels on contact with the gelling inducer at the outer surface of the droplet; and (d) forming a capsule including an interior core and a gel membrane by contacting the outer surface of the droplet with the gelling solution for a time sufficient for the gel forming polymer to form a gel membrane at the surface of the droplet to a desired thickness, the core containing the sperm sample.
A second method for providing sperm within a bead comprises the steps of (a) providing a sperm sample which is in a semen extender and a gel forming polymer; (b) forming the sperm sample into a droplet containing the sperm sample; (c) providing a gel inducing solution comprising an effective amount of an inducer which causes the gel forming polymer to gel on contact with the gelling inducer; and (d) forming a bead by contacting the droplet with the gel inducing solution for a time sufficient for the gel forming polymer to form a gel to provide the sperm sample in the bead.
In either method, it is preferable that the semen extender is substantially free of glucose and reactive oxygen species and provides an exogenous energy source which does not facilitate capacitation of the sperm in the absence of the reactive oxygen species or the glucose. In particular, wherein the exogenous energy source is selected from the group consisting of fructose, fructose-6-phosphate, pyruvate, lactate, and mixture thereof.
It is further preferable that the gel forming polymer be an ionotropic polymer and the gel inducer is a polyvalent ion. In particular, the polymer is selected from the group consisting of sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, gelatin, iragacanth gum, xanthan gum, and mixture thereof.
The present invention further provides a method for encapsulating sperm within a thermal gel membrane capsule comprising the steps of (a) providing a sperm sample which is in a semen extender and a gelling inducer; (b) forming the sperm sample into a droplet containing the sperm sample, the droplet having an outer surface; (c) providing a gelling solution comprising an effective amount of a gel forming polymer which polymer gels on contact with the gelling inducer at the outer surface portion of the droplet and a thermal gel forming polymer at a temperature sufficient to inhibit the gel forming polymer from gelling; (d) forming a capsule including an interior core and a gel membrane by contacting the outer surface of the droplet with the gelling solution for a time sufficient for the gel forming polymer to form a gel membrane at the surface of the droplet to a desired thickness, the core containing the sperm sample, and the membrane entrapping the thermal gel forming polymer; and (e) removing the gel forming polymer to produce the thermal gel membrane capsule.
Further still, the present invention provides a method for encapsulating the sperm in a thermal gel bead, the method comprising the steps of (a) providing a sperm sample which is in a semen extender containing a thermal gel forming polymer and gelling inducer at a temperature sufficient to inhibit the gel forming polymer from gelling; (b) forming the sperm sample into a droplet containing the sperm sample; (c) providing a gelling solution to lower the temperature; (d) forming a bead by contacting the droplet with the gelling solution for a time sufficient for the gel forming polymer to form a gel; and (e) allowing the thermal gel to solidify to produce the thermal gel bead containing the sperm in the semen extender.
Further still, the present invention provides a method for providing the sperm in a thermal gel bead, the method comprising the steps of (a) providing a sperm sample which is in a semen extender containing a thermal gel forming polymer and a gel forming polymer at a temperature sufficient to inhibit the gel forming polymer from gelling; (b) forming the sperm sample into a droplet containing the sperm sample; (c) providing a gel inducing solution comprising an effective amount of a gel inducer which on contact with the gel forming polymer causes the gel forming polymer to gel; (d) forming a bead by contacting the droplet with the gel inducer solution for a time sufficient for the gel forming polymer to form a gel; and (e) allowing the thermal gel to solidify to produce the thermal gel bead that contains the sperm in the semen extender and the gel forming polymer.
Further still, the present invention provides a method for encapsulating the sperm in a thermal gel bead, comprising the steps of (a) providing a sperm sample which is in a semen extender containing a thermal gel forming polymer at a temperature sufficient to inhibit the gel forming polymer from gelling; (b) forming the sperm sample into a droplet containing the sperm sample; and (c) forming the bead by chilling the droplet to a temperature that is below the gelling temperature of the thermal gel whereby the droplet becomes a solid which contains the sperm in the semen extender.
Preferably in the aforementioned, the semen extender is substantially free of glucose and reactive oxygen species and provides an exogenous energy source which does not facilitate capacitation of the sperm in the absence of the reactive oxygen species or the glucose. Preferred exogenous energy sources include those selected from the group consisting of fructose, fructose-6-phosphate, pyruvate, lactate, and mixture thereof. It is also preferable that the gel forming polymer is induced to gel by a polyvalent ion. Preferably, the gel forming polymer is an ionotropic polymer, which can be selected from the group consisting of sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, gelatin, iragacanth gum, xanthan gum, and mixture thereof. It is preferable that the thermal gel be a polymer that is a solid at room temperature and a liquid at body temperature. Preferred thermal gels include those selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, and mixture thereof.
The present invention further provides a method for artificial insemination comprising (a) providing an admixture of particles containing sperm in a semen extender which provides an energy source that does not facilitate capacitation of the sperm wherein the admixture comprises particles that vary in chemical property and diameter; and (b) introducing the admixture into a uterus. In one embodiment, the method comprises particles that are capsules comprising a core containing the sperm sample in a semen extender and a gel membrane encapsulating the core wherein the admixture comprises capsules having membranes of different thicknesses. In another embodiment, the particles are beads comprising the sperm sample in a semen extender and thermal gel.
Preferably, in the artificial insemination method, the semen extender is substantially free of glucose and reactive oxygen species and provides an exogenous energy source which does not capacitate the sperm in the absence of the reactive oxygen species or the glucose. In particular, wherein the exogenous energy source is selected from the group consisting of fructose, fructose-6-phosphate, pyruvate, lactate, and mixtures thereof and wherein glucose or precursors for reactive oxygen species are delivered with the particles containing the sperm during the artificial insemination. It one embodiment, the particles containing glucose or precursors for reactive oxygen species are delivered with the particles containing the sperm during the artificial insemination. It is preferable that the reactive oxygen species is produced by a mixture of xanthine and xanthine oxidase or H2O2.
In particular embodiments, the gel forming polymer is a thermal gel. Preferably, a thermal gel selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, and mixture thereof.
In embodiments comprising a membrane, it is preferable that the membrane have a thickness from 0.1 to 3.5 mm and containing therein sperm in a semen extender.
In particular embodiments, the gel forming polymer is induced to gel by a polyvalent ion. Preferably, the gel forming polymer is an ionotropic polymer. The polymer can be selected from the group consisting of sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, gelatin, iragacanth gum, xanthan gum, and mixture thereof.
The present invention further provides a particle for the timed release of sperm in a uterus comprising sperm in a semen extender which provides an energy source that does not facilitate capacitation of the sperm and a gel forming polymer which forms the particle.
In one embodiment, the particle is a capsule comprising an inner core containing the sperm in a semen extender and a gel forming polymer membrane encapsulating the core wherein chemical property and thickness of the gel membrane determines the time release of the sperm when the capsule is introduced into the uterus. Preferably, the gel forming polymer is selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, iragacanth gum, xanthan gum, and mixture thereof.
In another embodiment, the particle is a bead comprising the sperm in a semen extender and a gel forming polymer wherein chemical property and diameter of the bead determines the time release of the sperm when the bead is introduced into the uterus.
In either embodiment, the semen extender is substantially free of glucose and reactive oxygen species and provides an exogenous energy source which does not facilitate capacitation of the sperm in the absence of the reactive oxygen species or the glucose. It is preferable that the exogenous energy source be selected from the group consisting of fructose, fructose-6-phosphate, pyruvate, lactate, and mixture thereof.
In particular embodiments, the gel forming polymer is selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, sodium alginate, chitosan, guar gum, gum arabic, carrageenan, pectin, iragacanth gum, xanthan gum, and mixture thereof. In other embodiments, particularly in the bead embodiment, the gel forming polymer is a thermal gel. Preferably, a thermal gel selected from the group consisting of agarose, kappa-carrageenan, gelatin, agar, polyurethane-polyether polymers, and mixture thereof. In a further embodiment of the bead, the bead is encapsulated in a membrane comprising a gel forming polymer.
It is an object of the present invention to provide a method for encapsulating sperm in a particle, either a capsule or a bead wherein the sperm is maintained in a precapacitated or non-capacitated form. A further object of the present invention is to provide particles which allow for the timed release of the sperm when the particle is implanted in the uterus.
It is also an object of the present invention to provide a method to prolong the shelf life of semen, particularly the shelf life of boar semen and concomitantly to expand or widen the fertile period of the sperm cells in the female reproductive tract.
These and other objects will become increasingly apparent through the following detailed description of the invention and examples.